Figure 1.
G6 reduces Jak2 and STAT3 phosphorylation in T98G cells.
A) Expression of phospho-Jak2 in three glioblastoma cell lines. γ2A+Jak2 cell lysates were used as a Jak2-positive control. Shown is one of three representative blots. B) T98G cells were treated with the indicated concentrations of G6 for 24 hours and examined for Jak2 and STAT3 phosphorylation levels by Western blot. Shown is one of three representative blots for each protein. Quantification of the Jak2 (C) and STAT3 (D) phosphorylation levels (n = 3 for each). *, p<0.05 relative to DMSO control treated cells as determined by ANOVA.
Figure 2.
G6 treatment reduces T98G cell viability and clonogenic growth potential.
A) T98G cells were treated with increasing concentrations of G6 for 72 hours and cell viability was measured by MTS assay. Each point was measured in triplicate. Shown is one of three representative results. The 10 µM and 30 µM conditions were significantly lower (p<0.05) when compared to cells treated with DMSO alone as determined by ANOVA. B) T98G cells were treated with G6 for the indicated times and concentrations. Cell viability was then measured via MTS. Each point was measured in triplicate. Shown is one of three representative results. **, p<0.01 relative to DMSO treated cells as determined by ANOVA. C) T98G cells were treated with G6 for 24 hours at the indicated concentrations, washed, and seeded in 100 mm dishes. Cells were grown for nine additional days, stained with crystal violet, and colonies were then counted. Shown is the average of four representative results. *, p<0.05 relative to DMSO treated cells as determined by ANOVA.
Figure 3.
G6 reduces migratory and invasive potential of T98G cells.
A) Cells were treated with G6 for 24 hours at the indicated concentrations, washed, and a scratch was made. Cell migration was monitored 24 and 48 hours later. Shown is one of three representative results. B) Cells were treated with G6 for 24 hours at the indicated concentrations, washed, and plated in un-coated 24-well inserts. Cell migration was examined by crystal violet stain after 24 hours. Shown is one of three representative results. C) Cells were treated with G6 for 24 hours at the indicated concentrations and plated in matrigel-coated 24-well inserts. Cell invasion was examined by crystal violet stain after 48 hours. Shown is one of three representative results. D) Quantification (n = 3) of migrating cells. *, p<0.05 vs. DMSO control treated cells as determined by ANOVA. E) Quantification (n = 3) of invading cells. Scale bars represent either 100 µm (A) or 50 µm (B and C). *, p<0.05 vs. DMSO control treated cells as determined by ANOVA.
Figure 4.
Jak2 knockdown reduces the migratory and invasive potential of T98G cells.
A) Jak2 mRNA expression was examined by qRT-PCR. Each sample was measured in triplicate. Shown is one of three representative results. B) Jak2 protein expression was examined by Western blot. C) Quantification of Jak2 protein expression from three independent blots. D) Cells were plated in 24-well inserts and migration was monitored after 24 hours. E) Quantification of migrating cells from four independent experiments. F) Cells were plated in matrigel-coated 24-well inserts and invasion was monitored after 48 hours. G) Quantification of invading cells from four independent experiments. Scale bars represent either 50 µm (D and 10X F) or 100 µm (4X F) *, p<0.05 relative to scrambled shRNA as determined by Student's t test.
Figure 5.
G6 induces caspase-dependent apoptosis in T98G cells.
A) Cells were treated with G6 for 48 hours and apoptosis was examined by TUNEL immunofluorescence. Shown is one of three independent results. Scale bar = 50 µm. B) Quantification (n = 3) of the percentage of TUNEL-positive cells. *, p<0.05 vs. DMSO control treated cells as determined by ANOVA. C) Cells were treated for the indicated times with a 10 µM concentration of G6 and Caspase 3/7 activity was measured by a luminescent assay at the indicated times. Each point was measured in triplicate and shown is one of three independent experiments. A significant difference (*, p<0.05) was noted between the 72 hour time points as determined by ANOVA. D) Cells were treated with G6 for 48 hours and full length Caspase-3 expression was measured by Western blot. Shown is one of two representative blots.
Figure 6.
G6 reduces tumor volume in T98G xenografts and this is coincident with increased expression of BIM and BAX.
A) After establishment of the tumors, mice began receiving daily injections of either DMSO (n = 4) or 10 mg/kg G6 (n = 5) at Day 0. Tumor volumes are plotted as a function of both treatment condition and time. B) Average mRNA levels of various apoptotic markers taken from the tumors. *, p<0.05, **, p<0.01.
Figure 7.
Reduced Tumor Volumes Correlate with Decreased Levels of phospho-Jak2 and phospho-STAT3.
Shown are representative tumor sections, from the same nine mice described in Figure 6, after they were fixed, sectioned, and stained. For the H&E stained sections, mitotic cells are indicated by the green arrowheads. For pJak2, pSTAT3, Ki-67, and trichrome, the relative intensity of staining was quantified via relative pixel counting. Average pixel counts were then plotted as a function treatment group. The objective lens magnifications were either 50X (H&E) or 40X (pJak2, pSTAT3, Ki-67, and Trichrome). Scale bar = 50 µm. *, p<0.05, **, p<0.01.
Figure 8.
G6 Reduces the Levels of Vimentin Protein within T98G-derived Tumors.
A) Expression of vimentin levels from the same nine tumor samples described in Figure 6 were determined by Western blot. Also shown are the levels of β-actin which served as a loading control. B) Quantification of vimentin protein levels normalized to β-actin. C) Representative anti-vimentin immuno-histochemistry images showing decreased vimentin expression in the G6 treated tumors. Scale bars represent either 50 µm (top panels) or 10 µm (bottom panels). D) The intensity of vimentin staining was quantified via relative pixel counting and the average pixel counts were then plotted as a function treatment group. E) Tumor sections were subjected to anti-vimentin immune-fluorescence (red) and cell nuclei were visualized via DAPI staining (blue). In response to G6, the remaining vimentin protein formed some irregular perinuclear aggregates. The scale bars represent either 20 µm (large panels) or 5 µm (single inlet panel)*, p<0.05, **, p<0.01.